Pathways regulating cytoplasmic mRNA stability and translation are major determinants of protein abundance in mammalian cells. We are interested in processes by which proteins are assembled into ribonucleoprotein complexes in response to translational repression and, in turn, regulate mRNA stability and re-initiation of translation. Recently, there has been a great deal of research into the role of cellular granules containing silenced mRNAs in regulating translation and RNA turnover, and these structures have been implicated in the progression of neurodegenerative and other diseases. However, little is known about the pathways determining assembly of mRNAs into granules and the roles of specific proteins within these structures. To address these questions, we are isolating ribonucleoprotein complexes assembled on endogenously expressed hairpin-tagged RNAs. Using a system we have previously developed to provide an unbiased catalog of proteins assembled on particular RNAs, we are now identifying proteins associated with mRNAs in a manner dependent on translational repression. Our characterization of these proteins will focus on understanding the biochemical basis for specific assembly of RNA-binding proteins into translationally repressed mRNPs in addition to uncovering their cellular functions. To this end, we are using high-throughput RNAseq approaches to determine the transcriptome-wide occupancy of these proteins under distinct cellular conditions and to characterize changes in RNA translation and abundance in response to protein overexpression and/or depletion. This approach has led to the identification of candidate factors with prominent roles in controlling the stability of mRNAs encoding important gene expression regulators and the development of novel techniques for studying the relationship between translation and mRNA decay. First, we have found that a protein that we originally identified in association with translationally repressed mRNAs, the human zinc finger RNA-binding protein (ZFR), plays a major role in the regulation of the innate immune response. ZFR is required for the proper pre-mRNA splicing of mutually exclusive exons in the macroH2A histone variant gene (H2AFY). In the absence of ZFR, macroH2A mRNAs are subject to nonsense-mediated mRNA decay, abolishing expression of the histone variant. Moreover, we have found that ZFR regulates interferon beta transcription through macroH2A. In monocytes, ZFR expression is repressed through use of alternative transcription start sites, whereas macrophages induce ZFR, thereby repressing the interferon response. We are now investigating the regulation of ZFR activity and the mechanisms by which it influences pre-mRNA splicing. Second, we have extended the approach described above to develop a novel system for in vitro translation of endogenously assembled mRNAs. Following purification of PP7-tagged mRNAs, we incubate the purified complexes in translation-competent extracts. Strikingly, we find that this approach leads to much more efficient translation than is possible using traditional methods. Further, this translation is highly active under physiological buffer conditions, suggesting that mRNP assembly is important for the overall efficiency of translation. We have also shown that pharmacological manipulation of the in vitro translation system allows separation of distinct phases of translation on purified mRNPs. We are now developing this system for use as a general method to study the relationships among translation, RNP assembly, and RNA decay.